Neurotrophin proteins support survival and differentiation of developing neurons. Target tissues such as skin produce limited amounts of neurotrophins during critical periods of development that act to rescue neurons from programmed cell death. Though target-derived influences of nerve growth factor (NGF) have been well documented, effects of other neurotrophins are less clear.
During development of the vertebrate nervous system up to half of all neurons generated undergo a process of naturally occurring cell death (See Ard, M. D., Morest,
D. K. (1984),; Intl. J. Dev. Neurosci. 2, 535-547; and Oppenheim, R. W. (1991). Ann. Rev. Neurosci. 14, 453-501).
Neuronal death typically occurs shortly after neurons make functional connections within their target field. Survival during this period of innervation is thought to be dependent on the synthesis by the target tissue of a limited quantity of neurotropic substances. (See Thoenen, H. and Barde, Y. A. (1980), Physiol. Rev. 60, 1284-1334; Oppenheim, R. W. (1989), Trends in Neurosci. 12, 252-255; and Davies, A. M., Larmet, Y., Wright, E., Vogel, K. S. (1991), J. Cell Sci. 15, 111-116)).
The competition for target field-derived neurotrophic factors is thought to serve at least two functions: to ensure that an appropriate number of synaptic contacts are made at the target and to eliminate inappropriate neuronal projections. (See Oppenheim, R. W. (1981),. In Studies in Developmental Biology (ed. W. M. Cowan) p 74-133. Oxford University Press; and Cowan, W. M., Fawcett, J. W., O'Leary, D. D. M., and Stanfield, B. B. (1984), Science 225, 1258-1265)).
This concept is referred to as the neurotrophic hypothesis and predicts that the number of neurons in the adult could be increased if a higher concentration of neurotrophic factor(s) was available during the critical time when synaptic contacts are being established.
The NT-3 neurotrophin appears to have a role in neuron survival and maintenance. NT-3 is a member of the brain derived neurotrophic factor (BDNF)/nerve growth factor (NGF) neurotrophin-4 (NT-4) gene family. The development and maintenance of the nervous system depends on these proteins known as neurotrophic factors. Widespread neuronal cell death accompanies normal development of the central nervous system and peripheral nervous system, and plays a crucial role in regulating the number of neurons which project to a given target field (Berg, 1982, Neuronal Development, 397-331; Cowan et al., 1984, 225:1258-65).
Ablation and transplantation studies of peripheral target tissues during development have shown that neuronal cell death results from the competition among neurons for limiting amounts of survival factors (neurotrophic factors) produced in their projection fields. These observations led to the identification of nerve growth factor (NGF), which remains by far the best characterized neurotrophic molecule (Levi, Montalchini, and Angeletti, 1968, Physiol. Rev., 48:534-69; Thoenen and Barde, 1980, Rev. 60:1284-335).
Because NGF supports only a limited set of neuronal populations, the existence of additional neurotrophic factors has long been postulated (Varon, S. and Adler, R., 1981, Adv. Cellular Neurobiology, 2: 115-63; Barde et al., 1987, Prob. Brain Res., 71:185-9). While it is now clear that other neurotrophic factors exist, their extremely low abundance has impeded their molecular characterization. Nevertheless, purification of small amounts of two such proteins, namely brain derived neurotrophic factor and ciliary neurotrophic factor have recently permitted their cloning and sequencing (Leibrock et al., 1989, Nature, 341:149-52; Stockli et al., 1989, Nature, 342:21-28; and Lin et al., 1989, Science, 246:1023-25).
There have been numerous reports of neurotrophic factor activity in extracts of a great variety of tissues and in conditioned culture media of many different cell types. Progress in characterizing these activities and purifying the compounds has been hampered by the fact that such activities are present in extremely small amounts, the range of picograms and nanograms per gram of tissue.
Nerve growth factor (NGF) is a prototypical target-derived neurotrophic substance that has been shown to be essential for the survival and differentiation of neural crest-derived sensory neurons, sympathetic neurons, and forebrain cholinergic neurons. (See Levi-Montalcini, R. and Booker B. (1960), Proc. Natl. Acad. Sci. U.S.A 46, 384-391, and Hefti, F. (1986) J. Neurosci. 6, 2155-2161; Williams, L. R., Varon, S., Peterson, G., Wictorin, K., Fischer, W., Bjorklund, A. & Gage, F. H. (1986), Proc. Natl. Acad. Sci. U.S.A. 83, 9231-9235).
U.S. Pat. No. 5,180,820 to Barde et al. discloses nucleic acid sequences encoding brain derived neurotrophic factor of humans. U.S. Pat. No. 5,229,500 to Barde et al. claims pharmaceutical compositions including purified isolated nucleic acid sequences encoding brain derived neurotrophic factor.
U.S. Pat. No. 5,169,764 to Shooter et al. discloses and claims a nucleic acid molecule encoding a chimeric protein which has neurotrophic activity and consists of a chimeric protein including two neurotrophic factors selected from brain derived neurotrophic factor, ciliary neurotrophic factor, neurotrophin-3 and nerve growth factor.
U.S. Pat. No. 5,235,043 to Collins et al. discloses and claims a method for folding human mature nerve growth factor recombinantly expressed in E. coli wherein the protein maintains substantially full biological activity. U.S. Pat. No. 5,218,094 to de La Valle discloses a nominal neurotrophic factor isolated from mammalian brain tissue. U.S. Pat. No. 5,349,056 to Panayotatos discloses and claims a modified human ciliary neurotrophic factor. U.S. Pat. No. 5,364,769 to Rosenthal et al. discloses neurotrophic factor-4, isolated nucleotide sequences. U.S. Pat. No. 5,210,026 to Kovesdi et al. discloses human MK protein which is homologous to humor- and heparin-binding neurotrophic factor.
U.S. Pat. No. 5,272,063 to Chan et al. discloses and claims a method of producing biologically active mature human .beta.-nerve growth factor in insect cells.
International Publication WO 91/03568 to Hyman et al. discloses a derived neurotrophic factor and claims recombinant DNA molecule comprising a nucleic acid sequence encoding brain derived neurotrophic factor.
International Publication 91/03569 to Hohn discloses neurotrophin-3. The publication claims a recombinant DNA molecule comprising a nucleic acid sequence encoding neurotrophin-3 or subsequences thereof. This international publication is incorporated herein by reference in its entirety.
Various transgenic mice have been patented. U.S. Pat. No. 4,736,866 to Leder et al. discloses a transgenic non-human eukaryotic animal whose germ cells and somatic cells contain an activated oncogene sequence introduced into the animal or an ancestor of the animal at an embryonic stage. The embryo of the mice were microinjected with approximately 500 copies of the RSV-S107 c-myc plasmid. The injected eggs were transferred to pseudopregnant foster mothers and allowed to develop to term. The plasmid contains a Rous Sarcoma Virus enhancer and promoter sequence. The resultant mice showed expression of the c-myc gene in the salivary gland, spleen, testes, lung, brain, and preputial gland and intestinal tissue.
U.S. Pat. No. 5,175,383 to Leder et al. discloses a male transgenic mouse containing germ cells and somatic cells which contain a recombinant gene which is a vertebrate gene in the Int-2/FGF family which is capable of promoting benign prostatic hyperplasia or hypertrophy. The fusion gene which is injected into the mouse embryonic tissue comprises a promoter sequence controlling transcription of the recombinant gene such as a promoter derived from the mouse mammary tumor virus and cytomegalovirus. The recombinant gene is preferably substantially homologous with (i.e., greater than 50% homologous in terms of encoded amino acid sequence) a naturally occurring, vertebrate gene in the Int-2/FGF gene family of murine growth factor encoding genes or their vertebrate counterparts, including the murine acidic or basic fibroblast growth factor genes, the murine FGF-5 gene, the murine epidermal growth factor gene, the murine insulin-like growth factor-1 and -2 gene, the murine .alpha.-transforming growth factor gene with a murine hst/KS3 gene. The transgenic mice of Leder et al. exhibit prostate hyperplasia and give sterile offspring.
U.S. Pat. No. 5,087,571 to Leder et al. discloses a non-human eukaryotic animal whose germ cells and somatic cells contain an activated oncogene. The embryo of the mice were microinjected with approximately 500 copies of the RSV-S107 c-myc plasmid. The injected eggs were transferred to pseudopregnant foster mothers and allowed to develop to term. The plasmid contains a Rous Sarcoma Virus enhancer and promoter sequence. The resultant mice showed expression of the c-myc gene in the salivary gland, spleen, testes, lung, brain, and preputial gland and intestinal tissue.
U.S. Pat. No. 5,175,384 to Krimpenfort et al. discloses transgenic mice having a phenotype characterized by the substantial absence of mature T-lymphocytes. The mouse is produced by introducing a transgene into a zygote of a mouse which comprises gene fragment which encodes a T-cell antigen receptor polypeptide variant which is incapable of mediating T-cell maturation in the transgenic mouse.
U.S. Pat. No. 5,175,385 to Wagner et al. discloses the production of a transgenic mouse with enhanced viral resistance which is transmissible to its offspring. The transgenic mouse is prepared by introduction of a gene encoding a human interferon having anti-viral activity into a host mouse. The plasmid of Wagner et al. contains a methallothionein-1 promoter and the genomic human beta interferon gene. When these mice were injected with pseudorabies virus they showed an increased resistance to the virus and although many mice died they died considerably later than did the control animals.
Vassar et al., in the Proceedings of the National Academy of Sciences, Volume 86, pages 1563-1567, March 1989, disclose "Tissue-specific and Differentiation-specific Expression of a Human K14 Keratin Gene in Transgenic Mice". This publication discloses that Vassar et al. used a plasmid containing a K14 keratin promoter and a K14 keratin gene sequence tagged with a sequence from the neuropeptide substance P and determined the expression of the plasmid and K14 keratin tagged substance P in the skin of transgenic mice.
Vassar et al. in Gene & Development, Volume 5, pages 714-727, (1991) disclose "Transgenic Mice Provide New Insights into the Role of TGF-.alpha. During Epidermal Development and Differentiation". This publication discloses the construction of transgenic mice using a plasmid which contains the K14 keratin promoter and the TGF-.alpha. gene. Vassar et al. disclose that the TGF-.alpha. gene belongs to the epidermal growth factor family of proteins and shows structural homology with the epidermal growth factor. These transgenic mice showed gross phenotypic abnormalities in their skin including flaky outer epidermal layers with stunted hair growth and wrinkling. The plasmid used to create this transgenic mouse also included human growth hormone fusion gene.
U.S. Pat. No. 5,387,742 discloses transgenic mice which exhibit amyloid brain deposits characteristic of Alzheimer's disease.
PCT Publication WO 93/00909 is directed to a method of treating neurotrophin-expressing tumors by administering a sequence complementary to at least a portion of a RNA transcript of brain-derived neurotrophic factor gene.
Heretofore there has been no convenient method to investigate how neurotrophic factors such as NT-3, affect neuron survival and development of the peripheral nervous system or provide a practical model for testing drug candidates for the treatment of neurodegenerative disorders of the brain such as Parkinson's syndrome and Alzheimer's disease and assessing the effect of drug candidates on the central nervous system (CNS) and peripheral nervous system (PNS).
The present invention overcomes the deficiency by providing transgenic mice which express increased levels of Neurotrophin-3 in the epidermis and other stratified, keratinized epithelium. The transgene DNA construct injected into the mouse embryos contain a human K14 keratin promoter and enhancer region linked to the coding sequences of mouse Neurotrophin-3 gene. The mice can be used to study the role of NT-3 in supporting neuronal survival and in neurodegenerative disorders of the CNS and PNS.